This project will continue to examine the relationship between cardiac metabolism and myocardial contractility with special emphasis upon the influence of cyclic nucleotides (cyclic AMP and cyclic GMP) as regulators of myocardial metabolism and contractility. The cause and consequence of the oscillation in myocardial cyclic AMP during the contraction cycle, recently discovered in this project, will be carefully examined to determine the basic mechanism of this rapid rise and fall in cyclic AMP concentration. In addition, the influence of cardioactive drugs and ions will be studied to determine their influence on the cyclic AMP oscillation. Studies to determine the consequences of this transient rise and fall in cyclic AMP will be directed toward a search for oscillatory cyclic AMP dependent specific protein phosphorylation and dephosphorylation as a means to regulate myocardial calcium fluxes and cardiac contractility on a beat to beat basis. Methods already under development will be perfected to determine free and protein kinase bound cyclic nucleotides. A detailed study of the effects of hormonal stimulation in myocardium and other selected tissues on free and bound cyclic AMP will be examined to more accurately assess hormone action. Pools of cyclic nucleotides will be studied in myocardium using prelabeling techniques and specific activity determinations of the cyclic nucleotides and precursor trinucleotides. A further aspect of this project is to further the development of methodology capable of detecting most of the phosphorylated intermediates of metabolism. This technique will be refined using gradient high pressure anion exchange chromatography with differential continuous picomole sensitive U.V. and phosphate analysis of the chromatographic effluent. This technique will allow us to view hormone action on a broader basis and could be useful in detecting early changes in known or yet undiscovered metabolites as mediators of hormone and drug action.